Positional scanning, active compound

Illustration of positional scanning. Four sets of sublibraries are generated with one building block for every given position of the molecule defined. Screening reveals the prrferential building block for every single position. The active compound BCAD can be deduced and is synthesised for evaluation. 

Because of their ease of synthesis and their structural similarity to peptides, many laboratories have used peptoids as the basis for combinatorial drug discovery. Peptoids were among the first non-natural compounds used to establish the basic principles and practical methods of combinatorial discovery [17]. Typically, diverse libraries of relatively short peptoids (< 10 residues) are synthesized by the mix-and-split method and then screened for biological activity. Individual active compounds can then be identified by iterative re-synthesis, sequencing of compounds on individual beads, or indirect deduction by the preparation of positional scanning libraries. 

A hybrid of the one-bead-one-peptide and positional-scanning library formats (see Section 4.3.V.3.2.2) is presented by an approach termed library of libraries ,which is directed toward the identification of pharmacophore motifs, i.e. structural motifs necessary for the bioactivity of interest, rather than the complete structures of individual active compounds. This library format enables the identification of specific, i.e. nonreplaceable, positions of a peptide with a bioactivity of interest versus unspecific positions, which can be replaced by a variety of different amino acids without loss in activity. In the example presented,a hexapeptide library is generated as 160000 (20 x20) sublibraries (beads), which represent all possible combinations of three defined and three mixture positions within the hexapeptide framework, i.e. 8000 (20 ) different combinations of three amino acids at the defined positions within a given arrangement of the hexapeptide multiphed by 20 possible different arrangements of three defined and three mixture positions. While the defined positions are coupled using the DCR method,01 the mixture positions are introduced by the coupling of amino acid mixtures. 

A positional scanning combinatorial library (Fig. 3) can be used in place of iterative synthesis and screening [9]. In this approach, all library mixtures are synthesized and assayed for activity. The most active mixture from each positional library reveals the preferred residue at each position. Because all possible combinations of residues are represented in one of the sublibraries, the approach should be insensitive to synergistic effects associated with the structure. The orthogonal combinatorial library consists of two different libraries made up of the same compounds and synthesized in a way that mixtures from each library will only share one peptide or subunit [10]. In this way, these libraries are nonoverlapping, or orthogonal. 

A process called deconvolution is commonly employed to determine biological activity.The final library pools are not combined but are tested as either on-bead or detached compound mixtures. The most active pool defines which synthon is preferred in the last step. The synthesis is repeated to the penultimate set of pools and these are then allowed to react with the best last step synthon. Alternatively, pools of conserved resin from the penultimate step held back during the original synthesis may be used. The most active pool found on retesting defines the best last two synthons. This process is repeated until the most active member is identified. A somewhat similar method termed position scanning also has found successful application, especially in the analysis of peptides. ... 

Three deconvolution strategies can be used for the structural determination of the biologically most active compounds in a combinatorial library comprising mixtures of up to several thousand compounds (1) iterative deconvolution [47, 98] (2) deconvolution by positional scanning [99-101] or (3) deconvolution by orthogonal libraries [102-104]. 

However, if in one position there is no clear preference for a definitive substituent, all combinations of the preferred substituent must be synthesized in order to find the most active compound. Although active compounds are identified without iterative synthesis using positional scanning, in comparison with iterative deconvolution there is an increased likelihood that the most potent compound(s) will not be identified [110,111]. 

Extensive studies of the use of mixtures carried out by this laboratory and others have enabled the rapid, cost-effective identification of extremely active, highly specific individual compounds as reviewed in this chapter. There appears to be widespread skepticism that mixtures in combination with the positional scanning approach are effective in identifying active heterocycles, although they are widely accepted as methods for identifying peptides. However, these methods clearly are extremely effective and broadly applicable, as has been shown in this chapter and in many other studies. There is also nothing inherently unique about peptides or other oligomers that permits their successful use in these formats as compared with heterocycles. 

Positional scanning, a non-interative method for peptide library deconvolution. Positional scanning relies on the synthesis of partial compound libraries that represent first-order sub-libraries in which one position of the peptide sequence is kept invariant while aU other positions are varied. Once biological activity has been detected in the complete library, aU the sub-libraries are screened additionally in the same biological assay. Consequently, positional scatming reveals the optimum residue for every position in a peptide. 

There are several ways to deconvolute or identify the individual active compounds in an active mixture. These include iterative resynthesis and the synthesis of overlapping mixtures, such as the positional scanning method of Houghten et al." In iterative resynthesis, the most active pools are resynthesized as several smaller subpools with a variable position resolved. This process is repeated until every position has been resolved and individual active compounds have been made. In Houghten s method, the complete library is... 

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